Materials and Methods
This is a cross-sectional study which is conducted in Tongji Hospital, Tongji University School of Medicine, Shanghai, China, from January 2019 to December 2019. The newly diagnosed diabetic patients were enrolled into this study when they were diagnosed with type 2 diabetes for the first time according to the criteria by World Health Organization (1999), and the non-diabetic controls were enrolled from their age-matched community populations. A sample size calculation was conducted based on the primary marker in this study, bone alkaline phosphatase (BALP). We used the average BALP (12.96 ± 6.73 μg/L) of non-diabetic controls from a previous literature and the percentage of BALP changed in T2DM (22.5% increased) was determined according to another study. The sample size needed for each group is 84. All the participants in this study met the following inclusion and exclusion criteria. The inclusion criteria include the following: (1) age >45 years old; (2) postmenopausal women [serum follicle-stimulating hormone (FSH) >20 IU/L and/or age >55 years and no menses for at least a year]; and (3) never use any approved anti-diabetic drugs. The exclusion criteria include the following: (1) history of fractures in the past 6 months; (2) liver dysfunction (alanine aminotransferase >40 U/L or aspartate aminotransferase >40 U/L) or renal dysfunction (estimated glomerular filtration rate <90 ml/min/1.73 m2); (3) malignant tumour; and (4) having diseases and medicines that can affect bone metabolism such as rheumatoid arthritis, hyperparathyroidism, glucocorticoid, thyroid hormones, oestrogen, thiazolidinedione and anti-osteoporosis drugs. Ultimately, 88 patients with newly diagnosed T2DM and 152 non-diabetic control patients were included in this study after applying these criteria. This study was approved by the Ethics Committee of Tongji Hospital, Tongji University School of Medicine and following the ethical standards of the Declaration of Helsinki. All participants signed informed consent. This study has been registered in the Chinese Clinical Trials Registry (ID: ChiCTR1800020077).
Demographics Information and Clinical History. Baseline demographics information including age, weight, height, body mass index (BMI), time after menopause, status of current smoking, current drinking and clinical history were recorded. BMI (kg/m2) was calculated by dividing weight (kg) by the square of height (m2). The status of drinking was defined as drinking more than or equal to three units of alcohol per day (one unit of alcohol means 8g of alcohol).
Biochemical Parameter Measurement. Peripheral venous blood was collected after overnight fasting to measure the following indices: liver and renal function, lipid profile [total cholesterol (TCH), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL)], glycosylated haemoglobin (haemoglobin A1c, HbA1c), bone turnover markers and bone metabolism-related hormones [bone alkaline phosphatase (BALP), procollagen type I intact N-terminal (P1NP), osteocalcin (OC), tartrate-resistant acid phosphatase-5b (TRACP-5b), C-terminal cross-linking telopeptide of type I collagen (CTX), 25-hydroxyvitamin D and parathyroid hormone (PTH)], calcium (Ca), corrected calcium (corrected Ca) and phosphorus (P). HbA1c was detected by high-performance liquid chromatography using whole blood. Serum P1NP, OC, CTX, 25-hydroxyvitamin D [25(OH)D] and PTH were measured by electrochemiluminescence assay (Roche Diagnostics; coefficient of variation of intra- and inter-assay <10%). Serum BALP and TRACP-5b were measured by enzyme immunoassay (IDS Ltd; coefficient of variation of intra- and inter-assay <10%). Liver function, renal function, lipid profiles and electrolytes were detected by an automatic chemistry analyzer using serum. Corrected Ca(mmol/L) was calculated according to the following formula: Ca(mmol/L) + 0.02 × [40-albumin(g/L)]. All samples were collected within 7 days before or after the onset of T2DM when the patients were not taking anti-diabetic drugs.
Bone Mineral Density Detection. Bone mineral density (BMD) at lumbar spine 1–4, femur neck and total hip were detected by dual-energy X-ray absorptiometry (DXA, HOLOGIC Discovery; coefficient of variation <1%). Osteoporosis (OP) is diagnosed according to International Osteoporosis Foundation (IOF) guideline.
Statistical Analysis. Version 20.0 of Statistical Package for the Social Science (SPSS, IBM, Armonk, NY, USA) was used to conduct statistical analysis. The normality of the data was judged according to normality test. Continuous variables in normal distribution were expressed as mean±SD. And median (p25-p75) was used to describe continuous variables that showed skewed distribution. Categorical variables were reported as number and percentage. For comparison of continuous variables between the T2DM group and non-diabetic group, t test or the Mann-Whitney U test was performed depending on whether the data were in a normal distribution. And for comparison of categorical variables, the chi-square test was used. After logarithmic transformation for variables that did not satisfy normal distribution, the Pearson correlation was conducted to analyse whether each variable had an association with bone metabolism-related markers. The variables that showed possible associations (P <.1 according to the results of Pearson correlation) were taken into the multiple linear regression to adjust for potential confounding factors. P <.05 was considered to be statistically significant for all tests above, and all P-values were two-tailed.
Clin Endocrinol. 2021;95(3):430-438. © 2021 Blackwell Publishing